Cloning
Cloning is the process of creating an identical copy of an original. A clone in the biological sense, therefore, is a single cell (like bacteria, lymphocytes etc.) or multi-cellular organism that is genetically identical to another living organism. Sometimes this can refer to "natural" clones made either when an organism reproduces asexually or when two genetically identical individuals are produced by accident (as with identical twins), but in common parlance the clone is an identical copy by some conscious design. Also see clone (genetics). The term clone is derived from κλων, the Greek word for "twig". In horticulture, the spelling clon was used until the twentieth century; the final e'' came into use to indicate the vowel is a "long o" instead of a "short o". Since the term entered the popular lexicon in a more general context, the spelling ''clone has been used exclusively. Molecular cloning refers to the procedure where by a DNA sequence is amplified with the aid of genetic engineering techniques. Cloning is frequently employed to amplify DNA fragments containing genes, an essential step in their subsequent analysis. Frequently, the term cloning is misleadingly used to refer to the identification of the chromosomal location of a gene associated with a particular phenotype of interest. In practice, localisation of the gene does not always enable one to amplify the relevant genomic sequence. The beginnings of this research began with Devin Hale and Alex Bernot in a small laboratory in South Euclid, OH. Cloning of any DNA sequence comprises of the following four steps: amplification, ligation, transfection, and screening/selection. Initially, the DNA fragment of interest needs to be amplified (many copies need to be produced). Amplification is commonly achieved by means of PCR. Subsequently, a ligation procedure is employed whereby the amplified fragment is inserted into a vector. The vector (which is frequently circular) is linearised by means of restriction enzymes, and incubated with the fragment of interest under appropriate conditions that allow for ligation to occur. The yield of the ligation is routinely low and depends on the particular procedure employed. Following ligation the vector with the insert of interest is transfected to cells. Most commonly electroporation is employed, although a number of alternative techniques are available. Finally the transfected cells are cultured. As the aforementioned procedures are of particularly low yield, there is the need to identify the cell colonies that have been transfected with the construct of interest containing the desired insertion sequence. Modern cloning vectors include selectable antibiotic resistance markers, which allow only for cells in which the vector has been transfected to grow. However this selection step does not guarantee that the DNA insert is present in the vector. Further investigation of the resulting colonies is required to confirm that cloning was successful. This can be accomplished by means of blue/white screening and/or PCR, possibly followed by DNA sequencing. Cellular Cloning a cell means to derive a population of cells (a clonal population) from a single cell. This is an important in vitro procedure when the expansion of a single cell with certain characteristics is desired, for example in the production of gene-targeted ES cells. Most individuals began as a single cell (a zygote) and are therefore the result of clonal expansion in vivo. Cloning means to create a new organism with the same genetic information as a cell from an existing one. In a modern context, this can involve somatic cell nuclear transfer in which a cell of the organism to be cloned, with its nucleus containing the DNA, is transferred into an egg cell which has had its nucleus removed. As the nucleus contains almost all of the genetic information of a lifeform, the "host" egg cell will develop into an organism with genetically identical nuclear DNA to the nucleus "donor". However, this process does not conserve the mitochondrial genome (of the nucleus donor) unless the nucleus and egg cell donor were the same individual. Thus, nuclear transfer clones are not clones in the strictest sense because the mitochondrial genome is not the same as that of the nucleus donor cell from which it was produced. This may have important implications for cross-species nuclear transfer in which nuclear-mitochondrial incompatibilities may lead to inviability. The first animal clone was a frog, cloned by Thomas J. King and Robert W. Briggs in 1952. Horticultural The term clone is used in horticulture to mean all descendants of a single plant, produced by vegetative reproduction. Many horticultural varieties of plants are clones, having been derived from a single individual, multiplied by some process other than sexual reproduction. As an example, some European varieties of grapes represent clones that have been propagated for over two millennia. Other such examples are potatoes or bananas. Also grafting can be regarded as cloning, since all the shoots and branches coming from the graft are genetically a clone of a single individual. These are genuine examples of cloning in the broader biological sense, as they create genetically identical organisms by biological means, but this particular kind of cloning has not come under ethical scrutiny and is generally treated as an entirely different kind of operations. Natural clones Cloning exists in nature in some species and is referred to as parthenogenesis. An example is the "Little Fire Ant," Wasmannia auropunctata, which is native to Central and South America but has spread throughout many tropical environments."Bizarre stand-off in battle of the sexes" New Scientist, July 2 2005 In this species, circumstantial evidence from microsatellite DNA suggests that both queens and males may reproduce clonally in one population in Suriname. Species cloned The modern cloning techniques involving nuclear transfers have been successfully performed on several species. Land mark experiments in chronological order: * Tadpole: (1952) * Carp: (1963) In China, embryologist Tong Dizhou cloned a fish. He published the findings in an obscure Chinese science journal which was never translated into English. BLOODLINES. Timeline * Sheep: (1996) From early embryonic cells by Steen Willadsen. Megan and Morag cloned from differentiated embryonic cells in June 1995 and Dolly the sheep in 1997. * Rhesus Monkey: Tetra (female, January 2000) from embryo splitting * Cattle: Alpha and Beta (males, 2001) and (2005) Brazil Endangered cow cloned in Brazil * Cat: CopyCat "CC" (female, late 2001), Little Nicky, 2004, was the first cat cloned for commercial reasons * Mule: Idaho Gem, a john mule born May 4, 2003, was the first horse-family clone * Horse: Prometea, a Halflinger female born May 28, 2003, was the first horse clone * Human: embryo (2004) in Korea (retracted Verdict: Hwang's human stem cells were all fakes) and (2005) in Britain * Dog: Snuppy (2005) at the Seoul National University in South Korea. Dogs cloned from adult somatic cellsCloning Snuppy For a complete list see: List of animals that have been cloned. Ethical issues of cloning Roman Catholicism and many conservative Christian groups have opposed human cloning and the cloning of human embryos as they believe that from the moment of fertilization an embryo constitutes a person. Other Christian traditions like the United Church of Christ do not believe a fertilized ovum constitutes a person. Even traditions that do not believe that a fertilized ovum constitutes a person have opposed human embryo cloning. The World Council of Churches representing nearly 400 denominations world wide opposed cloning of both human embryos and whole humans in February 2006. The United Methodist Church opposed research and reproductive cloning in May 2000 and again in May 2004. Libertarian views on the subject suggest that it is in a person's constitutional rights to conduct this process, similar to abortion. Leon Kass said this about cloning, "Cloning represents a very clear, powerful, and immediate example in which we are in danger of turning procreation into manufacture." The main moral objection to attempts at human cloning right now is that a child might be damaged by such an origination. Researchers have not been able to safely and reliably create non-human primates by cloning. If there would come a day when the above safety objection was no more, the remaining ethical arguments against human cloning would be psychological (some would say, speculative) and religious ( a sense that such originations violate God's will or the natural scheme of things). Science fiction and popular movies uniformly present human cloning as a terrible thing for the person originated this way and for society. It has been a great taboo to think or argue otherwise. Under some conditions, however, say, if a person was safely born with a genotype that he or she later endorsed, human cloning could be morally permissible. Health aspects However, the success rate has been very low: Dolly the sheep was born after Ian Wilmut used 277 eggs to create 29 embryos, which only produced three lambs at birth, only one of which lived, Dolly. 70 calves have been created from 9,000 attempts and one third of them died young; Prometea took 328 attempts, and, more recently, Paris Texas was created after 400 attempts. Notably, although the first clones were frogs, no adult cloned frog has yet been produced from a somatic adult nucleus donor cell. There were early claims that Dolly had accelerated aging. Aging of this type is thought to be due to shortening of telomeres, regions at the tips of chromosomes which prevent genetic threads fraying every time a cell divides. Over time telomeres get worn down until cell-division is no longer possible - this is thought to be a cause of aging. However, subsequent studies showed that, if anything, Dolly's telomere were longer than normal. Dolly died in the year of 2003. Wilmut himself said that Dolly's early death had nothing to do with cloning but with a respiratory infection common to lambs like Dolly raised inside. Analysis of the cow's telomeres showed they had not only been 'reset' to birth-length, but they were actually longer - suggesting these clones would live longer life spans than normal cows (but many have died young after excessive growth). Researchers think that this could eventually be developed to reverse aging in humans, provided that this is based chiefly on shortening of telomeres. Although some work has been performed on telomeres and aging in nuclear transfer clones, the evidence is at an early stage. Therapeutic cloning is the procedure for creating stem cells genetically compatible with the patient. Therapeutic cloning might provide a way to grow organs in host carrier, which become completely compatible with the original. Host carrier growing poses a risk of trans-species diseases if the host is of a different species (e.g. a pig.) In human beings, this is a highly controversial issue, as it involves creating human embryos in vitro and then destroying them to obtain multipotent embryonic stem cells. Human cloning Human cloning is the creation of a genetically identical copy of an existing, or previously existing human or growing cloned tissue from that individual. The term is generally used to refer to artificial human cloning; human clones in the form of identical twins are commonplace, with their cloning occurring during the natural process of reproduction. Cloning extinct and endangered species Cloning, or more precisely, the reconstruction of functional DNA from extinct species has, for decades, been a dream of some scientists. The possible implications of this were dramatized in the novel by Michael Crichton and high budget Hollywood thriller Jurassic Park. In real life, one of the most anticipated targets for cloning was once the Woolly mammoth, but attempts to extract DNA from frozen mammoths have been unsuccessful, though a Japanese team is currently working toward this goal. In 2000, a cow named Bessie gave birth to a cloned Asian gaur, an endangered species, but the calf died after 2 days. In 2003, a banteng was successfully cloned, followed by three African wildcats from a thawed frozen embryo. These successes provided hope that similar techniques (using surrogate mothers of another species) might be used to clone extinct species. Anticipating this possibility, tissue samples from the last bucardo (Pyrenean Ibex), was frozen immediately after it died. Researchers are also considering cloning endangered species such as the giant panda, ocelot, and cheetah. Frozen Zoo at the San Diego Zoo now stores frozen tissue from the world's rarest and most endangered species. Scientists Close on Extinct Cloning, Heidi B. Perlman for washingtonpost.com, Oct. 8, 2000 "Cloning Endangered Species and Pets," Cloning After Dolly: Who's Still Afraid? Gregory Pence, Rowman & Littlefield, 2004. In 2002, geneticists at the Australian Museum announced that they had replicated DNA of the Thylacine (Tasmanian Tiger), extinct about 65 years previous, using polymerase chain reaction.Cloning to revive extinct species, Grant Holloway for cnn.com, May 28, 2002 However, on February 15 2005 the museum announced that it was stopping the project after tests showed the specimens' DNA had been too badly degraded by the (ethanol) preservative. Most recently, on May 15 2005, it was announced that the project would be revived, with new participation from researchers in New South Wales and Victoria. One of the continuing obstacles in the attempt to clone extinct species is the need for nearly perfect DNA. Cloning from a single specimen could not create a viable breeding population in sexually reproducing animals. Furthermore, even if males and females could be cloned, the question would remain open if they would be viable at all in the absence of parents that teach or show them natural behavior. Essentially, even if cloning an extinct species would succeed - it must be considered that cloning still is an experimental technology that succeeds only by chance - it is far more likely than not that any resulting animals, even if they were healthy, would be little more than curios or museum pieces. Cloning endangered species is a highly ideological issue. Many conservation biologists and environmentalists vehemently oppose cloning endagnered species not because they think it won't work but because they think it might deter donations to preserve habitat and wild populations. The rule-of-thumb in animal conservation is that, if it is still feasible to conserve habitat and viable wild populations, breeding in captivity should not be undertaken in isolation. In a review, Ehrenfeld (2006) concludes that cloning in animal conservation is an experimental technology that at its present state cannot be expected to work except by pure chance and utterly fails a cost-benefit analysis. Furthermore, it is likely to siphon funds from established and working projects and does not address any of the issues underlying animal extinction (such as habitat destruction, hunting or other overexploitation, and an impoverished gene pool). In plant conservation, cloning technologies are well-established and used on a regular base in conservation, but care must be taken to ensure genetic diversity. He concludes: "Vertebrate cloning poses little risk to the environment, but it can consume scarce conservation resources, and its chances of success in preserving species seem poor. To date, the conservation benefits of transgenics and vertebrate cloning remain entirely theoretical, but many of the risks are known and documented. Conservation biologists should devote their research and energies to the established methods of conservation, none of which require transgenics or vertebrate cloning." Dolly The Sheep Dolly (5 July 1996 – 14 February 2003), a ewe, was the first mammal to have been successfully cloned from an adult cell. She was cloned at the Roslin Institute in Scotland and lived there until her death when she was 6. Her birth was announced on 22 February 1997. The name "Dolly" came from a suggestion by Jesse Haase who helped with her birth, in honour of Dolly Parton, because it was a mammary cell that was cloned. The technique that was made famous by her birth is somatic cell nuclear transfer, in which a non-reproductive cell containing a nucleus is placed in a de-nucleated ovum (which then develops into a fetus). When Dolly was cloned in 1996 from a cell taken from a six-year-old ewe, she became the center of much controversy that still exists today. On 9 April 2003 her stuffed remains were placed at Edinburgh's Royal Museum, part of the National Museums of Scotland. Technical Hurdles Cloning is quite inefficient and usually there are over 600 to 1000 nuclear transfers before one is able to grow into a stem cell. This inefficiency is thought to be due to genetic imprinting in the cloned adult cell that interferes with the correct gene expression in the embryo. Even those animals that are successfully cloned are not as healthy as the original animal. For example, Dolly had arthritis and sign of premature aging. See methylation and epigenetic. See also * Christian views on cloning References *Pence, Gregory E. (1998): Who's Afraid of Human Cloning? Rowman & Littlefield. *Pence, Gregory E. (2004): Cloning After Dolly: Who's Still Afraid? Rowman & Littlefield. *Ehrenfeld, David (2006): Transgenics and Vertebrate Cloning as Tools for Species Conservation. Conservation Biology 20(3): 723-732. DOI:10.1111/j.1523-1739.2006.00399.x HTML abstract External links and references *The Reproductive Cloning Network. Cloning articles, resources and links *Cloning in Focus, an accessible and comprehensive look at cloning research from the University of Utah's Genetic Science Learning Center *Click and Clone. Try it yourself in the virtual mouse cloning laboratory, from the University of Utah's Genetic Science Learning Center *Cloning timeline: from CNN *CBC Digital Archives – Canada Enters the Clone Age Category:Cloning Category:Molecular biology da:Kloning de:Klonen eo:Klonado el:Κλωνοποίηση es:Clonación fi:Kloonaus fr:Clonage ia:Clonage id:Kloning it:Clonazione he:שיבוט ja:クローニング nl:Klonen pl:Klonowanie ru:Клонирование sv:Kloning tr:Klonlama ur:مثل تولید zh:克隆 {{enWP|